The present invention relates to a new method for evaluating the heat treatment to which a proteinic nutrient such as milk is subjected.
Currently, the specialist in the art has at his disposal some methods for evaluating the heat treatment of milk, two of which are official: the measurement of the lactulose in milks subjected to a U.H.T. process, on the one hand, and the measurement of the beta-lactoglobulin for pasteurised milks on the other hand. These two measurement techniques use high performance liquid chromatography (H.P.L.C.). The first of these methods allows the U.H.T. treatment to be controlled but with a time-lag of at least three hours, which prohibits any rapid intervention on the U.H.T. treatment parameters. The second allows one to appreciate the quality of a pasteurised milk, but likewise with too great a time-lag.
The implementation of these two processes does not allow the manufacturer to intervene rapidly on the current process: a certain literage of milk will therefore have undergone a heat treatment (U.H.T., pasteurisation) under poor conditions, before a correction to the process has been able to be made.
The specialist in the art likewise knows other methods, in particular for measuring the degradation of the proteins such as those present in heated milks. In particular, the products resulting from the Maillard reaction, such as furosine, are measured.
These methods of the prior art, the reliability of which cannot be questioned, present in particular three major disadvantages: as already explained above, they are slow and the results are only known after at least a period of three hours; they only allow a maximum of about twenty samples to be dosaged per day, which is hardly sufficient to follow a continuous process; and they are relatively costly.
Also, one of the aims of the present invention is to provide a method for evaluating the heat treatment to which a proteinic nutrient such as milk is subjected, which allows results to be obtained within short spaces of time compatible with the manufacturers"" requirements.
Another aim of the present invention is to provide such a method which permits the measurement of approximately one hundred samples per day.
A supplementary aim of the invention is to provide a method of this type which permits the evaluation of the effect of a heat treatment on the nutritional quality of the proteins of a nutrient such as as those of milk.
These aims, and also others which will be apparent hereinafter, are addressed by a method for evaluating the heat treatment to which a proteinic nutrient such as milk is subjected, which is characterised, according to the present invention, in that it comprises the following steps:
a) taking a sample of said nutrient and adding thereto an ionic strength buffer and of appropriate pH for obtaining on the one hand a precipitate of proteins denatured by the heat treatment and on the other hand a transparent supernatant containing proteins which are still soluble;
b) analysing the tryptophan present in the supernatant; and
c) analysing the fluorescent by-products derived from the advanced Maillard reaction present in the supernatant.
Preferably, the tryptophan present in the supernatant is measured by its fluorescence; for example, the measurement is carried out at an excitation wavelength of 290 nm and an emission wavelength of between 330 and 350 nm to obtain a maximum sensitivity. The measurement of the tryptophan could likewise be carried out by its U.V. absorption.
Advantageously, the measurement of the peptide fluorescent by-products derived from the advanced Maillard reaction present in the supernatant is realized with an excitation wavelength of 350 nm and emission wavelength of between 420 and 440 nm to obtain a maximum sensitivity.
So as to show better the conditions of implementation of a method in accordance with the present invention and also its advantages, the specialist in the art should refer to the examples below.
Beforehand, it will be recalled that as a function of the heat treatment, the proteins present in a proteinic nutrient such as milk undergo transformations which allow this heat treatment to be controlled.
In accordance with the present invention, on the one hand tryptophan was selected as marker of the level of denaturation of the proteins and, on the other hand, the fluorescent products derived from the advanced Maillard reaction, which alters the nutritional quality of the protein, as a reflection of the intensity of the reaction between the lysine and the products of carbonyl groups (s) such as sugars and peroxided lipids.